In humans and higher primates, fetal trophoblasts gain access to the lumens of dilated uterine capillaries and migrate along endothelium against the flow of blood eventually remodeling spiral arteries. Our general hypothesis is that migration of trophoblasts within uterine blood vessels is regulated by blood flow-derived shear stress. New data now show that trophoblasts migrate against flow when cultured on top of confluent uterine endothelial cells. To account for these observations, two, not mutually exclusive, mechanisms are proposed. First, we suggest that shear stress causes an asymmetric distribution of immobilized chemokines or adhesion molecules on endothelial cells that generates a haptotactic gradient directing trophoblast migration against flow. Second, we suggest that interaction of trophoblasts with immobilized chemokines and/or adhesion molecules on endothelial cells results in trophoblast activation causing trophoblasts to respond to shear stress by migrating against flow. Four aims will allow us to distinguish these mechanisms and characterize the features that define the shear stress-dependent migratory phenotype. First we will use videomicroscopy to characterize the effect of different levels of shear stress on the directional migration of trophoblasts on uterine endothelial cells. Aim 2 uses confocal microscopy to study the role of chemokines and endothelial adhesion molecules in shear stress-mediated trophoblast migration. We will also use migration checkerboard assays to determine whether chemokine- or adhesion molecule-induced trophoblast migration is haptotactic, chemotactic or chemokinetic. Aim 3 determines the effect of shear stress and trophoblast-endothelial interaction on induction of a migratory trophoblast phenotype. We will use confocal microscopy to characterize the leading edge/trailing edge distribution of CCR5, beta1 integrin, and cytoskeletal elements in trophoblasts exposed to different levels of shear stress in the presence of endothelial cells. Expression of these proteins will be quantiated by laser scanning cytometry, immunoblotting, and quantitative RT-PCR analysis. Function-blocking antibodies will identify the role of trophoblast adhesion molecules and chemokine receptors in flow-induced migration on endothelium. Aim 4 will use quantitative laser scanning cytometry to examine the expression of chemokine receptors, chemokines, and adhesion molecules in intravascular trophoblasts and endothelial cells in serial sections of uterine tissue.